title: Deletion of algK in mucoid Pseudomonas aeruginosa blocks alginate polymer formation and results in uronic acid secretion.
abstract: Chronic pulmonary infection with Pseudomonas aeruginosa is a common and serious problem in patients with cystic fibrosis (CF). The P. aeruginosa isolates from these patients typically have a mucoid colony morphology due to overproduction of the exopolysaccharide alginate, which contributes to the persistence of the organisms in the CF lung. Most of the alginate biosynthetic genes are clustered in the algD operon, located at 34 min on the chromosome. Alginate biosynthesis begins with the formation of an activated monomer, GDP-mannuronate, which is known to occur via the products of the algA, algC, and algD genes. Polymannuronate forms in the periplasm, but the gene products involved in mannuronate translocation across the inner membrane and its polymerization are not known. One locus of the operon which remained uncharacterized was a new gene called algK between alg44 and algE. We sequenced algK from the mucoid CF isolate FRD1 and expressed it in Escherichia coli, which revealed a polypeptide of the predicted size (52 kDa). The sequence of AlgK showed an apparent signal peptide characteristic of a lipoprotein. AlgK-PhoA fusion proteins were constructed and shown to be active, indicating that AlgK has a periplasmic subcellular localization. To test the phenotype of an AlgK-mutant, the algK coding sequence was replaced with a nonpolar gentamicin resistance cassette to avoid polar effects on genes downstream of algK that are essential for polymer formation. The algKdelta mutant was nonmucoid, demonstrating that AlgK was required for alginate production. Also, AlgK- mutants demonstrated a small-colony phenotype on L agar, suggesting that the loss of AlgK also caused a growth defect. The mutant phenotypes were complemented by a plasmid expressing algK in trans. When the algKdelta mutation was placed in an algJ::Tn501 background, where algA was not expressed due to polar transposon effects, the growth defect was not observed. AlgK- mutants appeared to accumulate a toxic extracellular product, and we hypothesized that this could be an unpolymerized alginate precursor. High levels of low-molecular-weight uronic acid were produced by the AlgK- mutant. When AlgK- culture supernatants were subjected to dialysis, high levels of uronic acids diffused out of the dialysis sac, and no uronic acids were detectable after extensive dialysis. In contrast, the mucoid wild-type strain produced only polymerized uronic acids (i.e., alginate), whereas the algKdelta algJ::Tn501 mutant produced no uronic acids. Thus, the alginate pathway in an AlgK- mutant was blocked after transport but at a step before polymerization, suggesting that AlgK plays an important role in the polymerization of mannuronate to alginate.
authors.affiliation: Department of Microbiology and Immunology, University of Tennessee and Veterans Affairs Medical Center, Memphis 38163, USA.
authors.forename: S
authors.initials: S
authors.lastname: Jain
authors.forename: D E
authors.initials: DE
authors.lastname: Ohman
mesh_terms: D000464:Alginates / Q000378:metabolism*; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D017353:Gene Deletion; D015870:Gene Expression; D020723:Glucuronic Acid; D006603:Hexuronic Acids; D008359:Mannose-6-Phosphate Isomerase / Q000235:genetics; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics; D016296:Mutagenesis; D009713:Nucleotidyltransferases / Q000235:genetics; D010641:Phenotype; D011108:Polymers; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D014574:Uronic Acids / Q000378:metabolism*
chemical_list: C104798:AlgK protein, Pseudomonas aeruginosa; D000464:Alginates; D001426:Bacterial Proteins; D006603:Hexuronic Acids; D009097:Multienzyme Complexes; D011108:Polymers; D014574:Uronic Acids; C080207:phosphomannose isomerase-guanosine diphospho-D-mannose pyrophosphorylase; D020723:Glucuronic Acid; D009713:Nucleotidyltransferases; D008359:Mannose-6-Phosphate Isomerase